The in vivo micronucleus test is a method devised primarily to screen chemicals for chromosomal breaking (clastogenic) activity (Schmid, W. (Mutation Res. 31, 9 (1975)), Salamone, et al., Morris (Mutation Res. 74, 347 (1980)), Heddle, J. A., et al. (Mutation Res. 123, 61(1983)), Salamone, M. F. and Heddle, J.A. Chemical Mutagens (F. J. de Serres, ed., Plenum Press) 8, 111 (1983)). The test is based on the observation that mitotic cells with chromatid breaks or chromatid exchanges exhibit disturbances in the anaphase distribution of their chromatin. After telophase, this displaced chromatin can be excluded from the nuclei of the daughter cells and is found in the cytoplasm as a micronucleus. Blood cells provide a sensitive model for evaluating clastogenic events since the nucleus of the erythrocyte stem cell is expelled a few hours after the last mitosis yielding DNA deficient cells. Treatment with clastogens or spindle positions which cause chromosomal breaks in the stem cell result in the formation of easily detectable micronuclei (MNs) in these anucleated young polychromatic erythrocytes (PCEs). These young anucleated cells are still rich in RNA and, therefore, exhibit unique staining patterns that distinguishes them from the mature normochromatic erythrocytes (RBCs). For example, when blood is stained with a metachromatic dye such as acridine orange (AO) (Hayashi, M., et al. (Mutation Res. 120, 241 (1983)), the DNA of a micronucleus exhibits a bright green-yellow fluorescence. In contrast the young RNA rich anucleated PCEs exhibit red fluorescence when stained with AO and excited with a 488 nm light source. The RNA rich polychromatic cells (PCEs) find their way into the blood stream and eventually complete their evolution to the RNA deficient and nonfluorescent normochromatic red blood cells- the mature RBCs. The brief existence of the PCE cells (about 48 hrs) has been used by practioners of the art to define the time frame for the conventional micronucleus assay by counting only MN in the PCE population. This invention offers a more flexible analysis timeframe which is not dependent upon the PCE population and allows for a choice of assay times ranging from hours to weeks. Although bone marrow was used in the original micronucleus assay, McGregor et al. (Environmental Mutagenesis 2,509 (1980)) demonstrated that the micronucleated PCEs and RBCs accumulate in peripheral blood of mice following treatment with a clastogen. Blood provides a good supply of test material for the micronucleus test. The spontaneous background level of aberrations in blood or bone marrow cells is usually quite low (i.e. about 2 MN/1000 PCEs). Clastogenic agents can cause an increase in the relative number of micronuclei present.
The micronucleus test provides a relatively rapid and sensitive indicator of both chromosomal aberrations and chromosomal loss that leads to numerical chromosome anomalies. However, the conventional method of carrying out the test with bone marrow cells has inherent limitations. Femurs are the usual source of bone marrow cells, and it has been necessary to use large numbers of animals (e.g. 50-60 mice per test substance; 10 mice/set) in order to overcome variations between animals. One set of mice must be used as a control to determine the spontaneous MN background level which is then used as a baseline for measuring a clastogenic response. This manual test involves the preparation of large numbers of slides, followed by hand scoring the number of micronuclei present in 1000 polychromatic cells. The scoring operations are subject to human errors aristing from the level of experience of each technician. Statistical errors also occur due to the relatively small number of cells that are processed in the manual scoring mode. Manually scoring the slides for a single test substance requires days of cell counting, resulting in considerable level of fatigue which can also lead to inaccuracies. With current state of the art, a conventional micronucleus test requires 8-12 weeks to complete, and it is both tedious and labor intensive. Because of the low number of cells scored (1000 PCEs) in a manual micronucleus assay, the statistics obtained are usually marginal. Practitioners in the art realize the need for improved sensitive methods for analyzing micronucleated cells. One possible method of automation is high speed flow cytometry (FCM).
In the FCM process, cells pass in single file through a laser beam where their fluorescence and light scatter characteristics are determined. Instead of scoring only 1000 cells as with manual methods, the flow cytometer is able to routinely process 2000-5000 cells/second and can analyze the number of micronucleated cells and polychromatic cells in more than 1,000,000 total cells in a few minutes. Once the experimental conditions are optimized, the statistics of the assay is related to the number of cells processed, and as a consequence greater accuracy in scoring chromosomal aberrations can be achieved. In 1982, Hutter and Stohr ( Histochemistry 75,353 (1982)) made an attempt to apply flow cytometric analysis to the micronucleus assay. Using bone marrow cells, along with a DNA-specific fluorochrome (DAPI; 4'-6'-diamidino-2-phenylindole) and a fluorescent protein stain (SR101; sulforhodamine 101), they identified flow cytometry patterns of putative micronucleated cells. However, their method was not able to discriminate normochromatic (mature) from polychromatic (young) erythrocytes and the flow cytometer cell counts did not correlate well with hand counts. The current state of the art is further defined by the fact that an Automated Micronucleus Scoring Workshop was held in November, 1988 at Miliptas, Calif., sponsered by the Environmental Mutagen Society to Evaluate the feasibility of automating the micronucleus assay, using flow cytometry or image analysis process, further demonstrating that a suitable flow cytometry based MN assay is not presently available. No definitive methods were forthcoming from this meeting, but the underlying difficulties of automated micronucleus scoring were addressed. Thus, although it is clear that a need exists for scoring larger numbers of micronulceated cells, the means for achieving automation is not presently known to Practitioners in the art.
The development of an FCM based micronucleus assay has been difficult becuase the process requires optimization of conditions in the biological, chemical, instrumentation and analysis disciplines. In order to be functional, the process must deal with the fact that micronucleated cells are present in blood or bone marrow samples as rare events. The micronuclei are small in comparison to the parent nucleus and cover a considerable range from 0.5 to about 2.mu.m in diameter depending upon the position of a chromosomal break. Similarly, PCE staining is an experimental variable, since the RNA of a PCE is degraded over time and the fluorescence yield of a PCE is related to its age. A functional FCM based micronucleus process must deal with the specific limitations and requirements of a flow cytometer. For example, the cells must be fixed in a way to minimize aggregation and permit the cells to pass through the laser beam in single file. Cell fixation is critical since it will influence the morphology and staining properties of cells, as well as the resolution of cell population during FCM analysis. Some cell fixation methods such as those that use glutaraldehyde or paraformaldehyde result in increased autofluorescence which can be undesirable for an FCM based micronucleous assay. The cell staining process must also be optimized because the amount of DNA in the micronucleus is quite low. Since the conventional assay evaluates the number of micronuclei per 1000 PCEs, differential staining of micronuclei and PCEs could be required. The dyes employed in the assay must also be chosen to provide maximum fluorescent signal, reflecting the DNA and/or RNA content of a cell, and must be able to absorb light at the wavelength provided by the laser beam(s). A means must also be provided for optimizing the signal output of a micronucleus by the use of suitable filters and gains on the acquisition photomultiplier tubes (PMTs), as well as a means for acquiring data for the cells of interest. The data must be processed in order to highlight any changes that might occur in the micronucleus and/or PCE population. Finally, the process should provide reproducible results which accurately reflect the content of micronucleated cells in each sample. The purpose of this invention is to disclose a process for effectively analyzing changes in the micronucleated cell populations in the blood and bone marrow cells caused by the action of clastogenic agents.